Clinical features, management, and prognosis of Bacillus cereus sepsis in premature neonates

This study aimed to investigate the clinical characteristics, management and prognosis of Bacillus cereus sepsis in premature neonates. The clinical information of 8 premature neonates with B cereus sepsis who were treated in Shanghai Children Hospital from January 2015 to December 2019 was retrospectively collected from the medical records and analyzed. The neurodevelopment related conditions were collected at follow up visits at corrected age of 6 months and 12 months. Five patients developed meningitis, and cerebral magnetic resonance image showed abnormal in 5 patients. After treatment with meropenem and vancomycin, 1 patient died, and 7 patients survived and were smoothly discharged. At follow up visits, 1 patient was diagnosed with hydrocephalus and showed severely delayed neurodevelopment, 2 patients had mild delayed neurodevelopment, and the neurodevelopment was basically normal in remaining 4 patients. B cereus infection can cause severe complications of central nervous system, and affect neurodevelopmental outcome. Antibiotic treatment with meropenem and vancomycin is proven to be effective. Refreshing the central catheters is helpful for the prevention of B cereus sepsis and cerebral magnetic resonance image may be employed for the prognosis assessment.


Introduction
Bacillus cereus is a type of Gram positive, aerobic facultative bacillus which is widely found in the environment. [1] B cereus is a spore forming and ubiquitous bacterium present in soil, food, insect larvae, almost all surfaces and human skin. [2,3] The B cereus group is comprised of 8 closely related species: B cereus, Bacillus cytotoxicus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus toyonensis, and Bacillus anthracis. [4][5][6] In adults, B cereus mainly causes gastrointestinal infection and has been the third most common cause of food poisoning. [7] Besides food poisoning, [8] B cereus can also cause focal or systemic infection, including sepsis, endophthalmitis, pneumonia, meningitis and encephalitis, especially in immunosuppressed patients, and delayed treatment often compromises the clinical outcome. Contaminated ventilator equipment, intravenous catheters and ventriculoperitoneal shunts have also been identified as routes of transmission in neonates with B cereus meningitis. [9,10] Neonatal sepsis is a serious disease which threatens the health and life of neonates. The incidence of neonatal sepsis is 4.5‰ to 9.7‰ among survived neonates [11] and increases as gestational age and body weight decrease. [12] The term and/ or premature neonates are susceptible to late-onset sepsis. Common pathogens for neonatal late-onset sepsis in China are Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Though B cereus is a rare cause of neonatal sepsis, it can also cause hemorrhagic meningoencephalitis which may severely damage the developing brain of the neonates. Recently, some studies have reported B cereus infection in premature neonates, but the neurological outcome of these patients is poorly understood. This study was to analyze the clinical characteristics and management of B cereus infection in 8 premature neonates as well as the prognosis at 6 and 12 months, and the effects of empirical antibiotic therapy on the clinical outcomes of B cereus infection were also explored.

Ethical statement
This study was approved by the Ethics Committee of Shanghai Children Hospital (2021R036-E01).

Participants
We screened preterm neonates with sepsis who were hospitalized in the Department of Neonatology, Shanghai Children Hospital from January 2015 to December 2019. Eight Medicine premature neonates were diagnosed with B cereus sepsis based on the clinical manifestations and results from blood culture (After disinfection, 1 mL of blood was collected for blood culture).

Data collection
The information about clinical features and treatments was retrospectively collected from the medical records, and the prognosis was evaluated at follow up visits.

Outcome
After discharge, we followed up the patients, the patients received Gesell Developmental Schedules scoring at 6 months of corrected age and 7 patients completed Ages & Stages Questionnaires (3rd Edition) (ASQ-3) at 12 months of corrected age.

General information
There were 6 males and 2 females. Cesarean section was noted in 4 patients and vaginal delivery in remaining 4 patients. The gestational age at birth was 29 to 35 +4 weeks; the birth weight ranged from 1060 g to 2330 g; the Apgar score at 5 minutes was 8'-10'; the age at the diagnosis of sepsis ranged from 4 days to 31 days; 5 patients (No 1, 2, 4, 6, and 7) had a history of premature prolonged rupture of membrane; 1 patient (No 3) had a record of maternal cervical incompetence; 2 patients (No 5 and 7) were smaller than gestational age, 1 patient (No 2) received anti-infective treatment before delivery because her mother had positive blood culture. The main clinical features are summarized in Table 1.

Clinical manifestations
Six patients (No 1, 3, 5, 6, 7, and 8) had fever with the highest body temperature at 38.2 o C to 38.8 o C; none developed hypothermia; 1 patient (No 3) presented with convulsions; 4 patients (No 1, 2, 4, and 6) developed apnea; 2 patients (No 2 and 7) had abdominal distension and feeding intolerance; 1 patient (No 3) refused feeding; 1 patient (No 2) was on non-peros due to suspicion of necrotizing enterocolitis. As shown in Table 2, after birth, 1 patient (No 1) received mechanical ventilation with continuous positive airway pressure for 3 days; 4 patients (No 5, 6, 7 and 8) had indwelling of peripherally inserted central catheter (PICC) for 7 to 27 days, and the PICC was removed after the sepsis resolved; 3 patients (No 1, 2, and 4) had peripheral venous catheter; 1 patient (No 3) had no peripheral venous catheter. All patients had positive blood culture for B cereus.
Based on blood culture results, vancomycin was administered at meningitis dosing. In 6 patients, the disease condition was improved after vancomycin treatment; 1 patient (No 2) was treated with meropenem and ampicillin; 1 patient (No 3) was initially treated with benzylpenicilin and ceftazidime and then with meropenem, but the neonate was non-responsive to treatment and died. The patients with clinical improvement received antibiotic therapy for 14 to 49 days (Table 1).

Laboratory examinations
In 2 patients (No 2 and 8), the White blood cell count and the neutrophil ratio increased; leukocytopenia was noted in 5 patients (No 1, 3, 4, 5, and 6); 2 patients (No 3 and 7) developed thrombocytopenia; all the patients had increases in C reactive protein and procalcitonin to different extents. According to CLSI standard, all B cereus strains were resistant to penicillin and compound trimethoprim, but sensitive to erythromycin, clindamycin, gentamicin, vancomycin and levofloxacin. All the patients received lumbar puncture; 5 patients had concomitant meningitis according to the cerebrospinal fluid (CSF) assay ( Table 3). The culture of all catheter tips failed to yield any bacterial growth.

Cerebral magnetic resonance image (MRI)
Six patients received cerebral MRI. As shown in Figure 1, MRI showed normal in 1 patient (No 5); 4 patients (No 1, 4, 6 and 7) had presentations of intracranial hemorrhage or leukomalacia; 1 patient (No 8) had meningeal enhancement on MRI; 1 patient (No 1) developed hydrocephalus on follow-up examination, and ventriculoperitoneal shunt operation was performed.

Outcomes
One patient died, and 7 patients survived and were discharged smoothly. All the survived patients received follow up through regular outpatient visit, the development of central nervous system was assessed and early intervention was administered if necessary. Four patients received Gesell Developmental Schedules scoring at 6 months of corrected age and all the patients completed Ages & Stages Questionnaires (3rd Edition) (ASQ-3) at 12 months of corrected age. Results showed 1 patient (No 1) had severely delayed neurodevelopment and underwent rehabilitation treatment, 2 patients (No 5 and 6) had mildly delayed neurodevelopment, and the neurodevelopment was basically normal in remaining 4 patients (Table 4).

Discussion
B cereus has been found in a variety of environmental reservoirs such as ventilator equipment, intravascular catheters and linen.
In addition to food poisoning, B cereus may also cause systemic or focal infections in immunologically compromised or immunocompetent individuals. The spectrum of infections includes fulminant bacteremia, central nervous system infection (meningitis and brain abscess) and other complications. [1] The pathogenicity of B cereus is closely related to the production of tissue damaging/reactive exoenzymes. The secretin includes 4 kinds of hemolysins, 3 distinct phospholipases, an emesis-inducing toxin, and 3 pore-forming enterotoxin: hemolysin BL, nonhemolytic enterotoxins and cytotoxin K.

Clinical manifestations
The immune function of premature infants is not mature, and the incidence of sepsis is high. The clinical manifestations were atypical. B cereus is not a common bacterium associated with neonatal sepsis. Studies have reported that the majority of strains (41%) are isolated from neonates, 3/4 of whom were premature infants with low birth weight. [13] The virulence test of bacterial strains has indicated that the average production of bacterial toxin from neonates is low, suggesting that neonates are particularly sensitive to B cereus infection, even the strains with low toxin production, or some other unknown factors may be responsible for the infection in neonates. Neonates are more susceptible to B cereus infection than adult, especially in those with catheter indwelling. In our study, all the neonates were preterm and had venous catheter indwelling after birth, and half of them had PICC. In our cases, the culture of all catheter tips failed to identify any bacteria. However, catheter indwelling is a risk factor of B cereus infection. Thus, we recommend that once B cereus infection is confirmed in children, the catheters should be removed as soon as possible, and the catheters may be indwelt again once the infection is controlled after effective antibiotic therapy. In addition, B cereus can cause severe late-onset hemorrhagic meningoencephalitis in preterm infants. In our study, we found that the clinical manifestations of these 8 patients were more like those of gram-negative sepsis. 6 patients had fever, 4 patients developed apnea, 1 patient presented with convulsions. These signs can also be initial signs of neonatal meningitis, especially convulsion.

Clinical examinations
According to the evaluation for neonatal sepsis, [14] for newborns with sepsis, especially those with central nervous system symptoms, the lumbar puncture should be further improved. In our study, all patients received lumbar puncture; 5/8 neonates had signs and symptoms of meningitis (CSF assay showed leukocytosis, low glucose, and increased protein). Lequin et al found B cereus could cause a severe late-onset hemorrhagic meningoencephalitis in preterm infants, the ultrasound and MR images showed a typical pattern of mainly hemorrhagic and early cavitating, selective white matter destruction. [15] According to the patient symptoms and the results of lumbar puncture, 6/8 patients received cerebral MRI examination, among whom 5 had abnormal findings on cerebral MRI. 1 patient had normal CSF assay but abnormal findings on MRI. We, therefore, believe that lumbar puncture and cerebral MRI are necessary for preterm infants with B cereus septicemia.

Clinical treatments
Some studies have reported that most B cereus strains are resistant to penicillin and cephalosporin, but sensitive to aminoglycoside, carbapenem, vancomycin and chloramphenicol. [16] In Table 2 Time of mechanical ventilation and catheter indwelling.  our study, the patient was initially treated with benzylpenicilin and ceftazidime and then with meropenem, but the neonate was non-responsive to treatment and died. The other 7 patients were first treated with meropenem for empirical anti-infective therapy, which was effective, and vancomycin or ampicillin sulbactam were added according to the results of drug susceptibility tests. Our results showed B cereus strains were responsive to meropenem or vancomycin.

Outcomes
Preterm infants themselves are at risk for poor nervous system development, and sepsis and encephalopathy can increase the risk of stunting. [17] The B cereus septicemia in preterm infants is often serious and high mortality, and these patients are more likely to have neurological sequelae. Our study showed the prognosis was acceptable in 4 out of 7 surviving preterm infants (4/7) after active intervention, which was largely related to the close follow-up and good compliance to treatments. In the follow-up period, clinicians should also pay more attention to the development of the nervous system in the later stage, and timely intervention should be carried out for premature infants with B cereus septicemia if necessary.

Limitations and experience
There were still limitations in this study. In our study, only PICC tips were sampled, followed by bacterial culture, but breast milk, formula milk, sheets and other substances were not sampled for bacterial culture. Thus, the source of infection  Of note, more studies with large sample size are warranted to confirm our findings.

Conclusions
Positive blood culture of B cereus in premature neonates should not be deemed as contamination. B cereus infection can cause severe central nervous system complications. Lumbar puncture should be performed and timely anti-infective treatment is usually indispensible. Meningitis dosing meropenem and vancomycin are often effective in the treatment of B cereus infection. Cerebral MRI is needed before the antibiotic treatment is discontinued. Clinicians should pay attention to the neurological development at late stage for preterm infants with B cereus septicemia.
Author contributions